Báo cáo khoa học: SAF-3, a novel splice variant of the SAF-1/MAZ/Pur-1 family, is expressed during inflammation pptx

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SAF-3, a novel splice variant of the SAF-1/MAZ/Pur-1family, is expressed during inflammationAlpana Ray1, Srijita Dhar1, Arvind Shakya1, Papiya Ray1, Yasunori Okada2and Bimal K. Ray11 Department of Veterinary Pathobiology, University of Missouri, Columbia, MO, USA2 Department of Pathology, School of Medicine, Keio University, Tokyo, JapanIntroductionTranscription factors play a central role in regulatingcell growth and development as well as in cellularmaintenance as a result of their indispensable role insynthesizing mRNA. Dysregulation of transcriptionfactor activity leads to alteration in target gene expres-sion patterns, which is one of the most importantcauses of disease development and progression. Exten-sive studies on the characterization of transcriptionfactors have indicated that, in general, transcriptionfactors exist as a family of structurally related proteins,containing conserved and unique domains. The familymembers can perform similar tasks due to the con-served domains but may be functionally specific due tothe unique domains. The various members of theKeywordsgene expression; inflammation; SAF-1/MAZ/Pur-1; splice variant; transcription factorCorrespondenceB. K. Ray, Department of VeterinaryPathobiology, University of Missouri, 124Connaway Hall, Columbia, MO 65211, USAFax: +1 573 884 5414Tel: +1 573 882 4461E-mail: rayb@missouri.eduA. Ray, Department of VeterinaryPathobiology, University of Missouri, 126Connaway Hall, Columbia, MO 65211, USAFax: +1 573 884 5414Tel: +1 573 882 6728E-mail: rayal@missouri.eduDatabaseThe sequence for MAZ genomic DNA hasbeen submitted to the Genbank databaseunder the accession numbers D89880.(Received 6 January 2009, revised 12 May2009, accepted 5 June 2009)doi:10.1111/j.1742-4658.2009.07136.xThe Cys2His2-type zinc finger transcription factor serum amyloid A activa-ting factor 1 [SAF-1, also known as MAZ (myc-associated zinc fingerprotein) or Pur-1 (purine binding factor-1)] plays an important role in regu-lation of a variety of inflammation-responsive genes. An SAF-2 splice vari-ant acting as a negative regulator of SAF-1 was identified previously, andthe present study reports the identification of a novel SAF-3 splice variantthat is expressed during inflammation. SAF-3 mRNA, isolated from acDNA library produced from IL-1b-induced cells, originates from a previ-ously unknown first coding exon, and thereby contains a unique N-termi-nal domain but shares the same six zinc finger DNA-binding domains aspresent in SAF-1. In addition, a negatively functioning domain present atthe N-terminus of SAF-1 and SAF-2 is spliced out in SAF-3. The expres-sion of SAF-3 is very low in normal tissues and in cells grown undernormal conditions. However, RT-PCR analysis of mRNAs from cytokineand growth factor-induced cells as well of mRNAs isolated from severaldiseased tissues revealed abundant expression of SAF-3. The transactiva-tion potential of SAF-3 is much greater than that of the predominantlyexpressed splice variant SAF-1. These findings show that transcriptionalregulation of downstream inflammation-responsive genes by SAF/MAZ/Pur-1 is likely to be more complex than previously assumed. In addition,we show that SAF-3 expression initiates from an upstream novel promoter.This is the first report of the existence of multiple promoters regulatingexpression of the SAF/MAZ/Pur-1 family of proteins.AbbreviationsCAT, chloramphenicol acetyl transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAZ, myc-associated zinc finger protein;MMP, matrix metalloproteinase; OA, osteoarthritis; Pur-1, purine binding factor-1; RA, rheumatoid arthritis; SAF-1, serum amyloid Aactivating factor; VEGF, vascular endothelial growth factor.4276 FEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBSfamily are often generated from a single gene by alter-native splicing, which is recognized as an efficientmeans of increasing the diversity of proteins.Serum amyloid A activating factor 1 (SAF-1) is thefirst identified member of a transcription factor familycontaining multiple Cys2His2-type zinc finger proteins[1]. The human and mouse orthologs of SAF-1 areknown as myc-associated zinc finger protein (MAZ) [2]and purine binding factor-1 (Pur-1) [3], respectively.The SAF-1/MAZ/Pur-1 transcription factor is aninflammation-responsive protein, and regulates theexpression of a variety of genes that include serumamyloid A [1,4], vascular endothelial growth factor(VEGF) [5], p21 [6], several matrix metalloproteinases(MMPs) [7–10], c-myc [2], insulin [3], and the serotonin1A receptor [11], which are involved in diverse cellularprocesses and various pathogenic conditions. A num-ber of inflammatory stimuli such as cytokines [12],phorbol 12-myristate 13-acetate [13], lipopolysaccha-ride [14] and oxidized low-density lipoproteins [15]have been shown to activate SAF-1 protein andincrease its DNA-binding and transactivating func-tions. The SAF-1 DNA-binding and transcriptionalactivity is significantly increased in response to media-tors of signal transduction and phosphorylation by anumber of protein kinases [13,16,17]. The transcriptlevel of SAF-1/MAZ is also reported to be increasedin response to cytokine stimulation [18], in hepatocel-lular carcinoma [19], chronic myelogenous leukemia[20] and acute myeloid leukemia [21], and during skele-tal myocyte differentiation [22].In a previous analysis, we identified SAF-2 [23], thesecond member of this family, which is encoded by thesame gene by alternate splicing. Insertion of a newexon originating from the non-coding sequences ofintron 4 of the SAF-1/MAZ gene resulted in creationof a different C-terminus consisting of eight zinc fingerdomains in SAF-2 [23]. The SAF-2 isoform has amuch higher DNA-binding activity and acts as a nega-tive regulator of SAF-1 function under normal condi-tions [23]. During inflammation, SAF-2 expression isdown-regulated, which alleviates the repression ofSAF-1 activity and further promotes SAF-1-mediatedtransactivation of the target genes [23]. In this paper,we present evidence for a third member of SAF family,which is also transcribed from the same gene but origi-nates from an upstream novel start site and containsan entirely different N-terminus. Expression of SAF-3is restricted to inflammatory conditions. Further, wepresent evidence that SAF-3 is much more transcrip-tionally active than SAF-1. These results shed light onthe relevance of the generation of multiple distinctfunctional SAF isoforms, and imply the existence ofcombinatorial mechanisms that allow fine regulationby SAF-regulated genes.ResultsIdentification and characterization of a novel SAFisoformScreening of an IL-1b-induced human HTB-94 chon-drocyte cell cDNA library identified a novel humanSAF-1/MAZ/Pur-1 isoform that contains uniqueN-terminal sequences (Fig. 1). This clone, with anopen reading frame of 455 amino acids, was designatedSAF-3 (GenBank accession number FJ532357), withSAF-1/MAZ/Pur-1 being the originally identified iso-form [1–3]. Comparison of amino acid sequences indi-cated that the N-terminal region of SAF-3 is differentfrom that of SAF-1, after which both cDNAs containidentical sequences. The previously isolated SAF-2 iso-form differs from SAF-3 at both the N- and C-termini[23]. SAF-1 and SAF-2 have identical N-termini, butthe SAF-2 mRNA contains an unique exon near the 3¢end, and thus its C-terminus is different from that ofSAF-1 [23]. The open reading frames in SAF-1, SAF-2and SAF-3 code for 477, 493 and 455 amino acids,respectively.SAF-3 is produced by alternative splicingTo determine the presence of unique N-terminaldomain in SAF-3, we examined the genomic DNAsequence of human SAF-1/MAZ/Pur-1 [24]. Sequenceanalysis indicated that the unique N-terminal aminoacids of SAF-3 are encoded by a previously unidenti-fied exon (exon 1A) that is present 351 nucleotidesupstream of the first exon of human SAF-1/MAZ(Fig. 2A). The first exon in human SAF-3 (exon 1A)encodes 12 amino acids, including the initiator methio-nine (Fig. 2B). The nucleotide sequence around the ini-tiation ATG codon in SAF-3 matches the translationinitiation site consensus sequence as determined byKozak [25] (Fig. 2B). The second exon of SAF-3 (exon1C) starts at around the middle of the first exon ofSAF-1/SAF-2. The SAF-3 transcript also represents anin-frame splicing event, for which the open readingframe remains unchanged. The 5¢ and 3¢ splice junc-tions of exons 1A and 1C match consensus splicedonor and acceptor sequences (Fig. 2C and Table 1).The N-terminal amino acid sequences of three SAFisoforms are shown in Fig. 2D. We performed primerextension analysis to determine whether the SAF-3cDNA contains a full-length 5¢ UTR. A32P-end-labeled, 18-base antisense oligonucleotide primer wasA. Ray et al. Transcription factor SAF-3 is expressed during inflammationFEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBS 4277Fig. 1. Analysis of a structurally alteredform of SAF. The nucleic acid and predictedamino acid sequences of the cDNA encod-ing SAF-3 are shown. The initiator ATGcodon and stop codon are indicated.Transcription factor SAF-3 is expressed during inflammation A. Ray et al.4278 FEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBSutilized for the primer extension reaction, andindicated several possible transcription start sites forSAF-3 mRNA (Fig. 2E, lane 2). However, the esti-mated length of the major primer extension productcorresponded well with the sequence of cloned SAF-3,suggesting that this cDNA possibly contains a full-length 5¢ UTR. The two faint but longer primer-extended products that were visible in Fig. 2E, lane 2,probably arise from other transcription start sites, andare minor transcription products. Nested RT-PCRanalysis was performed for further verification of theexistence of novel exon 1A in SAF-3. A product of theA BE F G H CDFig. 2. Schematic representations of SAF splice variants. (A) Exons are indicated by white boxes, and 5¢ and 3¢ UTRs by speckled boxes.The introns and UTRs are not drawn to scale. The positions of the primers used for RT-PCR are indicated by bold lines. (B) Sequence ofexon 1A in SAF-3. The Kozak consensus sequence is indicated by a line above the sequence. (C) The exon/intron boundaries conform toconsensus splice junction sequences. (D) N-terminal amino acids of the SAF-1/SAF-2 and SAF-3 isoforms. (E) Primer extension analysis. A 5¢end-labeled oligonucleotide complementary to a sequence near the 5¢ end of sequenced cDNA (+4 to +21 with respect to translation initiatorATG codon) was used to prime cDNA synthesis using poly(A)+RNA from IL-1b-induced HTB-94 cells (lane 2). The arrow indicates the majorprimer extension product, and the arrowheads show minor primer extension products. As a negative control, yeast tRNA was used in sepa-rate extension reaction, and no extension product was produced (lane 3). Lane 1 contains a G-specific reaction of an unrelated sequence thatwas used as a size marker to determine the length of the primer extension products. (F) RT-PCR analysis. Total RNA isolated from IL-1b -induced HTB-94 cells was subjected to reverse transcription and nested PCR using SAF-3-specific (lane 1) and SAF-1-specific (lane 2) oligo-nucleotide primers. The amplified product was verified by direct DNA sequencing. (G) Bacterially expressed SAF-1 (lane 2) and SAF-3 (lane3) proteins were fractionated by SDS–PAGE. The migration positions of these proteins in lanes 2 and 3 are indicated. Lane 1 contains pro-teins from vector-transfected cells. (H) In vitro transcription and translation. A linear plasmid containing full-length SAF-3 cDNA downstreamof T7 RNA polymerase transcription start sequences was subjected to in vitro transcription and translation. The protein productswere labeled with35S-methionine, fractionated by SDS–PAGE and autoradiographed. Lane 1 contains no added DNA and lane 2 containsSAF-3 plasmid DNA. The sizes of the protein products were identified using standard protein molecular weight markers.Table 1. Sequences of exon/intron junctions in human SAF-3. Exonsequences are shown in upper-case letters, and intron sequencesare shown in lower-case letters.ExonExonsize(nt) 5¢ splice donorIntronsize(nt) 3¢ splice acceptor1A 75 ATCTTCCAGgtaacaac 625 cacctcagGGTCACGCC1C 87 CCATTCCAGgtgagtag 84 ctccgcagGCCGCGCCG2 851 CTTCTCCCGgtgtgcac 403 gtccccagGCCGGATCA364AATGTGAGgtaggaag 277 ctcctcagAAATGTGAG4 172 CAACAAAGgtacatgc 1335 ctgtgcagGTACTGGTG5 1028A. Ray et al. Transcription factor SAF-3 is expressed during inflammationFEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBS 4279right size was amplified from mRNAs of IL-1b-induced HTB-94 cells using an upstream primer corre-sponding to the 5¢ untranslated sequences of SAF-3and a downstream primer corresponding to thesequences at exon 2 (Fig. 2F, lane 1). The identity ofthe PCR-amplified product was verified by DNAsequence analysis. The same downstream primer atexon 2, together with an upstream primer correspond-ing to the SAF-1 sequence at exon 1B, produced anSAF-1-specific PCR product (Fig. 2F, lane 2). Thetranslation product of SAF-3 cDNA was determinedby cloning SAF-3 cDNA in a bacterial expression vec-tor (Fig. 2G). In line with the cDNA size, bacteriallyexpressed SAF-3 protein migrates slightly faster thanbacterially expressed SAF-1 protein in the same vector(Fig. 2G, compare lanes 3 and 2). We performed acoupled in vitro transcription and translation reactionto further determine whether SAF-3 is indeed initiatedfrom the first ATG codon. The SAF-3 protein trans-lated from the predicted ATG codon is 455 aminoacids long. The next ATG codon in the SAF-3 cDNAis 603 nucleotides downstream of the first ATG codon,and a protein initiated from this ATG would be ofconsiderable smaller size, containing 254 amino acidsand with an approximate molecular mass of 28 kDa.As seen in Fig. 2H, lane 3, the in vitro transcribed andtranslated protein product from SAF-3 cDNA was ofsimilar size to that obtained using the bacterialexpression system, indicating that the major translationproduct of SAF-3 mRNA is 455 amino acids long.Inflammation-responsive expression of SAF-3To determine the expression pattern of SAF-3, wehybridized a multiple-tissue Northern blot with aradiolabeled SAF-3-specific oligonucleotide probe (cor-responding to exon 1A), and found no detectablesignal (Fig. 3A). However, upon re-hybridization witha full-length SAF-1 cDNA probe containing sequencescommon to SAF-1, SAF-2 and SAF-3, the same blotshowed the presence of multiple bands (Fig. 3B). As apositive control for the SAF-3-specific oligonucleotideprobe, we prepared SAF-1 and SAF-3 RNA by in vitrotranscription and hybridized the RNA with radioactiveSAF-3 oligonucleotide probe. This probe clearlydetected in vitro transcribed SAF-3 mRNA but did notdetect SAF-1 mRNA (Fig. 3C). Together, these resultssuggest very low SAF-3 expression in normal tissues.Given that SAF-3 was isolated from a cDNA libraryproduced from IL-1b-induced cells, we examined thestatus of this isoform during cytokine stimulation ofcells. RT-PCR of RNA isolated from untreated andIL-1b-treated HTB-94 cells showed expression of adetectable level of SAF-3 only upon cytokine inductionA B C D EFig. 3. Cytokine or growth factor treatment stimulates expression of SAF-3. (A) Northern analysis of an RNA blot (Clontech) containing 1.0 lgof poly(A)+RNA per lane from various tissues as indicated. The blot was hybridized using a32P-labeled oligonucleotide probe containingunique exon 1A sequences of SAF-3 mRNA. (B) The same blot was stripped and re-hybridized with a full-length 1.4 kb32P-labeled SAF-1cDNA probe. (C) SAF-1 (lane 2) and SAF-3 (lane 3) RNAs were in vitro transcribed from corresponding cDNA plasmids by T7 RNA polymerase.Reaction products were fractionated in a 1% agarose gel, transferred to nylon membrane, and hybridized with a32P-labeled oligonucleotideprobe containing unique exon 1A sequences of SAF-3 mRNA. Lane 1 contains HindIII-digested kcI857 DNA. (D) HTB-94 and Saos-2 cellswere treated with or without IL-1b (500 UÆmL)1) or TGFb (5 ngÆmL)1), as indicated. Total RNA isolated from these cells was subjected toreverse transcription followed by nested PCR amplification to monitor SAF-3 expression. The same sets of RNAs were also used to monitorMMP-9 and GAPDH expression, as indicated. The PCR products were separated in a 1.5% agarose gel and visualized by ethidium bromidestaining. (E) Western blotting with SAF-3-specific antibody. One microgram each of purified bacterially expressed SAF-1 protein (lane 1) andSAF-3 protein (lane 2) and 50.0 lg each of uninduced (lane 3) and IL-1b-induced (lane 4) HTB-94 cell extracts were fractionated by 11% SDS–PAGE, transferred onto membrane and Western blotted with anti-SAF-3 serum. The arrow indicates SAF-3 protein in IL-1b-induced cells.Transcription factor SAF-3 is expressed during inflammation A. Ray et al.4280 FEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBS(Fig. 3D, lanes 1 and 2). SAF-3 was also detected inTGFb-induced Saos-2 osteosarcoma cells (Fig. 3D,lanes 3 and 4). As positive control, we examined theexpression of MMP-9, which is known to be inducedin HTB-94 cells by both IL-1b and TGFb. MMP-9expression was detected in both IL-1b- and TGFb-stimulated cells. As a control for the same input ofRNA in RT-PCR, the glyceraldehyde-3-phosphatedehydrogenase (GADPH) expression pattern was mon-itored, and this remained unchanged during cytokineinduction. To detect in vivo expression of SAF-3, a spe-cific antibody was generated utilizing the unique N-ter-minal peptide sequences. Western blot analysis wasperformed to confirm the specificity of this antibody, asit did not detect bacterially expressed SAF-1 proteinbut clearly detected bacterially expressed SAF-3 protein(Fig. 3E, lanes 1 and 2). SAF-3 expression was detectedat a low level in IL-1b-induced HTB-94 cells using thisantibody (Fig. 3E, lanes 3 and 4).Detection of SAF-3 mRNA in chronicinflammatory diseased tissuesGiven the cytokine-responsive expression of SAF-3, weexamined its level in diseased tissues. Osteoarthritis(OA) is a chronic inflammatory disease that involvesdegeneration of the cartilage tissue, while rheumatoidarthritis (RA) is a systematic chronic inflammatoryand destructive arthropathy. As seen in Fig. 4, SAF-3mRNA expression was detected in OA synovial(Fig. 4, lanes 3–6) and RA synovial (Fig. 4, lanes 7–9)tissues, but very little to no SAF-3 mRNA expressionwas detected in normal synovium (Fig. 4, lanes 1 and2). SAF-1 expression was detected in normal tissuesand was slightly elevated in both disease conditions.This result is consistent with previous findings indicat-ing that the main mode of activation of SAF-1 is bypost-translational modification, including phosphoryla-tion [13, 16, 17]. Together, these results indicated thatSAF-3 expression is very low and highly regulatedunder normal conditions, but increases in response topathogenic signals and cytokine or growth factorstimulation.SAF-3 is a superior transcriptional activatorSAF-3 contains six zinc finger motifs, and should havethe ability to interact with DNA at a similar level toSAF-1 [26]. However, because it contains a differentN-terminus, the transactivation potential of SAF-3may be different, and it may thereby regulate expres-sion of downstream genes at a different level. Todetermine the functional significance of SAF-3, wecompared its transactivation potential with that ofSAF-1. The SAF-3 expression plasmid transactivatedexpression of the SAF-3X-CAT reporter at a muchhigher level than the same amount of SAF-1 expressionplasmid DNA (Fig. 5A). To rule out the possibilitythat SAF-1 and SAF-3 proteins were not expressed atthe same level, we performed a Western blot assayusing an anti-His tag IgG and representative trans-fected cells (Fig. 5B). This experiment showed nodiscrepancy in the expression of proteins, indicatingthat SAF-3 is a superior transcriptional activatorcompared to SAF-1. For further verification, wecompared ability of these two isoforms to transactivateexpression of VEGF, a natural SAF-regulated gene [5].In correlation with previous results, the SAF-3expression plasmid increased expression of the 1.2VEGF-CAT reporter in a more effective manner(Fig. 5C). Together, these results show that the SAF-3splice variant has significantly higher transactivationpotential.SAF-3 mRNA is transcribed from a distincttranscription start siteAn SAF-1/SAF-2-specific 5¢ RACE did not reveal anyupstream 5¢ sequences in SAF-3 (data not shown); thissuggests that SAF-3 and SAF-1/SAF-2 mRNAs maybe transcribed from distinct promoter regions. Todetermine whether SAF-3 and SAF-1/SAF-2 mRNAsare initiated from different promoters, we examinedthe respective 5¢ flanking regions. Genomic DNAsequences upstream of SAF-3 and SAF-1/SAF-2 wereligated into the promoterless vector pBLCAT3 toproduce ()2000/+200)SAF-CAT, ()2000/)351)SAF-CAT and ()351/+200)SAF-CAT reporter constructs.Transient transfection of HTB-94 cells with thesereporters resulted in significantly higher levels ofchloramphenicol acetyl transferase (CAT) activity,Fig. 4. SAF-3 expression is detected in human arthritic tissues.Total RNA was isolated from representative normal, OA and RAsynovium tissues and subjected to RT-PCR using SAF-3-, SAF-1-and GAPDH-specific primers, as indicated. GAPDH expression wasused as an internal control.A. Ray et al. Transcription factor SAF-3 is expressed during inflammationFEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBS 4281albeit variable, compared to cells transfected with thepromoterless pBLCAT3 vector (Fig. 6). These resultsclearly indicate that the DNA sequences between)2000 and )351 and )351 and +200 contain neces-sary elements that can promote transcription. We con-clude from these results that the human SAF-1 genehas at least two promoters.DiscussionIn this paper, we describe a novel splice variant ofSAF-1/MAZ/Pur-1 family of transcription factors thatmay be involved in regulating inflammation-inducedexpression of various SAF targets associated withpathogenic conditions. In addition, we provide the firstevidence of the existence of two promoters in thehuman SAF-1/MAZ/Pur-1 gene that permit transcrip-tion of multiple mRNAs with different N-termini. Thenovel SAF-3 splice variant reported here is transcribedfrom the upstream promoter. SAF-3 is predominantlyexpressed in cytokine- and growth factor-treated cellsand in diseased tissues, but is barely detectable undernormal conditions.ABCFig. 5. Transactivation potential of SAF-3. (A) HTB-94 cells wereco-transfected with SAF3X-CAT2 reporter plasmid (0.5 lg) andpCMV-bgal expression plasmid (0.4 lg) without ()) or togetherwith (+) empty vector pcDNA3 (0.5 lg), pcDSAF-1 (0.5 lg) or pcD-SAF-3 (0.5 lg) expression plasmid DNA, as indicated. After 24 h,cells were harvested and equivalent amounts of cell extracts wereassayed for CAT reporter activity. The data shown represent themean ± SEM of three separate experiments (*P < 0.05 versuscontrol). (B) Western blot analysis of transfected cells with anti-Histag IgG. (C) CAT reporter assay. HTB-94 cells were transfectedwith 1.2VEGF-CAT reporter plasmid (0.5 lg) and pCMV-bgalexpression plasmid (0.4 lg) without ()) or together with increasingconcentrations (0.2, 0.3, 0.4 and 0.5 lg) of pcDSAF-1 or pcDSAF-3 expression plasmid DNA, as indicated. After 24 h, cells wereharvested and equivalent amount of cell extracts were assayed forCAT reporter activity. The data shown represent the mean ± SEMof three separate experiments (*P < 0.05).A B Fig. 6. SAF-3 mRNA is transcribed from an upstream promoter. (A)Schematic drawing of the two promoter regions in the SAF gene.(B) CAT reporter assay. Each of the reporter constructs (0.5 lg)was co-transfected together with pCMV-bgal expression plasmid(0.4 lg) into HTB-94 cells. After 24 h, cells were harvested andequivalent amounts of cell extracts were assayed for CAT reporteractivity. The data shown represent the mean ± SEM of threeseparate experiments (*P < 0.05).Transcription factor SAF-3 is expressed during inflammation A. Ray et al.4282 FEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBSAlternative splicing is a widespread mechanism ofgene regulation, and is also an efficient means ofincreasing the diversity of proteins from a single gene[27–29]. This versatile mode of gene regulation is uti-lized during development, sex determination, hormonalregulation and apoptosis. Deletion or inclusion of anexon during splicing can generate a family of transcrip-tion factors that may have subtle or dramatically dif-ferent properties. Due to such changes in specificityand/or binding strength, one member can act as a neg-ative regulator of another member. In the SAF familyof Cys2/His2-type zinc transcription factors, the threemembers that have been identified so far are generatedfrom a single gene by alternative splicing. SAF-1 andSAF-2 mRNAs initiate from the same start siteand thereby have identical N-termini but differentC-termini due to insertion of an exon in SAF-2 [23].The SAF-3 mRNA is transcribed from an upstreampromoter and contains a totally different N-terminus.In addition, a portion of the first exon constituting theN-terminus of SAF-1/SAF-2 mRNA is deleted inSAF-3 mRNA (Figs 1 and 2). In previous studies, thisregion of SAF-1/SAF-2 was shown to contain a nega-tively functioning module [26]. The SAF-2 isoformactivates SAF-1 under inflammatory conditions in aunique fashion [23]. Under normal conditions, SAF-2negatively regulates SAF-1 transactivation, but, asSAF-2 is down-regulated under various inflammatoryconditions, the repression of SAF-1 activity is relieved,which permits a further increase in the expression ofSAF-1 targets. In contrast to SAF-2, SAF-3 is specifi-cally expressed during inflammation. SAF-3 thusappears to play a significant role in the pathogenicconditions associated with increased expression ofmany SAF target genes, due to the combination ofinflammation-responsive expression of SAF-3 and thesuperior transactivation potential of SAF-3. Theunderlying mechanism for the increased transcriptionalfunction of SAF-3 is presently unknown. Increasedtranscriptional activity of SAF-3 could result from(a) a transactivating module present in the uniqueN-terminus, (b) binding of the N-terminus by ancillaryfactors, (c) the lack of a negatively functioning modulethat is present in the N-terminus of SAF-1 [26], or (d)all of the above.It was interesting to note the lack of a consensusTATA box and/or CAAT box in both promoters ofthe SAF gene. In this regard, SAF-1/MAZ/Pur-1resembles about the third of eukaryotic gene promot-ers that do not contain a consensus TATA box.Another notable feature of the two SAF promoters isthe presence of a high frequency of CpG dinucleotides,which are also known as CpG islands. The CpGislands have been shown to play an important role inepigenic control during mammalian development, andare frequently altered in many disease conditions suchas cancer [30–32]. In addition, methylation of thecytosines of CpG islands in the promoter or the firstexon has been shown to affect the rate of transcription[33], and ever since the clear demonstration of a causalrelationship between hypermethylation of the promoterof tumor suppressor genes and the development ofcancer, it has been believed that transcription of manygenes is repressed via DNA methylation [34]. Itremains to be investigated whether transcription fromthe upstream SAF promoter, i.e. expression of SAF-3mRNA, is regulated via DNA methylation.In conclusion, we show that the human SAF-1/MAZ/Pur-1 gene has two promoters, which areutilized to produce multiple mRNAs with unique prop-erties. A specific increase in the expression level ofSAF-3 transcript transcribed from the upstream pro-moter may determine the level of SAF protein duringinflammation and pathogenic conditions. Further anal-yses of the factors that modulate transcriptional activ-ity of the upstream SAF promoter are necessary toclarify the mechanisms regulating increased expressionof SAF-3 during inflammatory conditions.Experimental proceduresIsolation, cloning and sequencing of the SAF-3splice variantA kgt-11 cDNA library was prepared using mRNAs iso-lated from IL-1b-induced human HTB-94 cells. The librarywas screened using an SAF-1 cDNA probe. The DNAinserts from the selected clones were sub-cloned in pTZ19Uand sequenced. One of these cDNA clones contained theSAF-3 sequence. The promoter region of SAF-1/SAF-2/SAF-3 was isolated by screening a human genomic DNAlibrary in kEMBL3 (Clontech Laboratories Inc., MountainView, CA, USA) with a full-length SAF-1 cDNA probe.Three independent positive clones were selected. Regions ofthe phage DNA spanning the human SAF gene weresequenced.Cell cultures and transfectionHuman HTB-94 chondrocyte cells, derived from a primarygrade II chondrosarcoma, and human osteosarcoma Saos-2cells were cultured in Dulbecco’s modified Eagle’s mediumcontaining high glucose, 100 unitsÆmL)1penicillin and100 unitsÆmL)1streptomycin supplemented with 7% fetalcalf serum. Both cell lines were obtained from the Ameri-can Type Culture Collection, Manassas, VA, USA).A. Ray et al. Transcription factor SAF-3 is expressed during inflammationFEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBS 4283Transfection assays were performed as described previously[7]. pSV-b galactosidase (Promega Corporation, Madison,WI, USA) plasmid DNA was used as an internal control,and was assayed as described previously [35]. Cells wereharvested 24 h post-transfection, and CAT activity wasassayed as described previously [35]. All transfectionexperiments were performed at least three times.Plasmid constructsThe SAF3X-CAT2 reporter was constructed by ligatingthree tandem copies of SAF DNA-binding elements intothe pBLCAT2 vector as described previously [23]. 1.2VEG-F-CAT was constructed by ligating a 1.2 kb promoterDNA fragment of human VEGF gene into the pBLCAT3vector as described previously [5]. Expression plasmidspcDHis-SAF-3 and pcDHis-SAF-1 were constructed byligating full-length SAF-3 or SAF-1 cDNA into thepCDNA3.1-His vector (Invitrogen, Carlsbad, CA, USA).The ()2000/+200)SAF-CAT, ()2000/)351)SAF-CAT and()351/+200)SAF-CAT plasmids were prepared by PCRamplification of the respective DNA fragments of the pro-moter region of human SAF gene followed by ligation intothe pBLCAT3 plasmid vector.Preparation of SAF-1 and SAF-3 proteinsFor bacterially expressed SAF-1 and SAF-3 proteins, thecorresponding cDNAs were subcloned into the pRSETvector (Invitrogen). Proteins expressed from these con-structs were purified by nickel–agarose column chroma-tography (Invitrogen) according to the manufacturer’sprotocol.Isolation of RNA and RT-PCRTotal RNA was isolated from untreated HTB-94 cells andfrom HTB-94 and Saos-2 cells treated with IL-1b(500 UÆmL)1) and TGF-b (5 ngÆmL)1), respectively, usingthe guanidinium thiocyanate method [36]. Total RNA wasisolated from synovial tissue of OA and RA patients asdescribed previously [37]. Briefly, arthritic synovial tissuewas obtained from patients undergoing total knee joint orhip replacement. Synovial tissues were prepared from sevenRA knee joints and 14 OA knee joints. Informed consentwas obtained from the patients according to ethical guide-lines. RT-PCR was performed using an RT-PCR kitaccording to the manufacturer’s protocol (Invitrogen).DNase-treated RNA (1 lg) was used in the reverse tran-scription with random hexamers and oligo(dT)12–18as theextension primer. PCR was performed by denaturing at94 °C for 2 min, followed by incubation at 94 °C for 15 sand 68 °C for 1 min for 40 cycles. For detection of SAF-3mRNA, nested PCR was performed. In the first PCR, theSAF-3-specific forward primer 5¢-CGCGAGCCACCTCCCTCCCTCC-3¢ and reverse primer 5¢-GCTTCAGGGCCGCTGTGTCCAC-3¢ were used, which produced a345 bp product. The reaction products were separated in anagarose gel, and a portion of the first PCR-amplified prod-uct (345 bp) was punched out using a Pasteur pipette andresuspended in 200 lL of sterile dH2O; 1.0 lL of thissuspension was used as the template for the second PCR.The SAF-3 primers for the second PCR primers were5¢-CCGCCATGGAT CCCAGCAACTGGAGCAGC-3¢(forward) and 5¢-GAGAACCGGGAGCAAGTCCAC-3¢(reverse). The amplification product of the second PCR was208 bp. The reaction products were resolved in a 1.5% aga-rose gel, and the identity of amplified DNA was verified byDNA sequence analysis. The primers for the SAF-1-specificPCR were 5¢-CCATGTTCCCCGTGTTCCCTTGCACGCTG-3¢ (forward) and 5¢-GAGAACCGGGAGCAAGTCCAC-3¢ (reverse), and the amplification product was271 bp. The primers for MMP-9 were 5¢-GGCTCTCCAAGAAGCTTTTCTC-3¢ (forward; present in exon 10) and5¢-CATAGCTCACGTAGCCCACTTGG-3¢ (reverse; pres-ent in exon 13), and the amplification product was 378 bp.The primers for GAPDH were 5¢-TGCACCACCAACTGCTTAG-3¢ (forward) and 5¢-AGAGGCAGGGATGATGTTC-3¢ (reverse), and the amplification product was 177 bp.Northern blot analysisA multiple-tissue Northern blot (Clontech LaboratoriesInc.) was hybridized using a32P-labeled oligonucleotideprobe that contained the unique region of SAF-3. Thesequence of the SAF-3 specific oligonucleotide probe is5¢-CCAGGGTGAGCGCGAGCCACCTCCCTCCCTCCCTCCGCCATGGATCCCAGCAACTGGAGCAGCTTCATCTTCCAG-3¢. After stripping off the probe, the samemembrane was re-hybridized with a32P-labeled SAF-1cDNA probe containing the entire coding region (approxi-mately 1.4 kb).Primer extension analysisFor primer extension analysis, 0.5 lg of poly(A)+RNAfrom IL-1b-induced HTB-94 cells was hybridized with a 5¢-end32P-labeled, 18-base antisense oligonucleotide primer(5¢-GCTCCAGTTGCTGGGATC-3¢;106countsÆmin)1)corresponding to positions +4 to +21 with respect to theATG start codon of the SAF-3 cDNA. The probe andRNA were heated at 90 °C for 15 min in 80% formamide,40 mm Pipes pH 6.4, 400 mm NaCl, 1 mm EDTA buffer,and then incubated overnight at 50 °C. The annealedmRNA and oligonucleotide was ethanol-precipitated andresuspended in 50 lLof50mm Tris/HCl pH 8.3, 50 mmKCl, 10 m m MgCl2,1mm dithiothreitol, 0.5 mm spermi-dine, 1 mm each of the four dNTPs, 1000 UÆmL)1RNasinTranscription factor SAF-3 is expressed during inflammation A. Ray et al.4284 FEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBS(Promega) and 1000 UÆmL)1AMV reverse transcriptase(Promega), and incubated at 42 °C for 90 min. Thesample was extracted with phenol/chloroform and ethanol-precipitated. As a negative control, yeast tRNA was usedas the template in a separate extension reaction. The prod-ucts of primer extension reactions were fractionated in a12% polyacrylamide/urea sequencing gel.Preparation of SAF-3 and SAF-1 RNA by in vitrotranscriptionTo prepare SAF-3 and SAF-1 RNA transcripts, full-lengthSAF-3 and SAF-1 cDNAs cloned into pTZ19U vector weresubjected to in vitro transcription using T7 RNA polymer-ase and a commercial Riboprobe System T7 kit (Promega)according to the manufacturer’s protocol. The productswere fractionated in a 1% agarose gel, transferred to nylonmembrane and hybridized with radioactive SAF-3-specificoligonucleotide probe.In vitro transcription and translation of SAF-3proteinFull-length SAF-3 cDNA cloned into pTZ19U vector wassubjected to in vitro transcription and translation using aTNT T7 coupled reticulocyte lysate system kit (Promega)according tot the manufacturer’s protocol. In vitro tran-scription of SAF-3 mRNA was performed by using T7RNA polymerase, and transcribed SAF-3 mRNA was fur-ther in vitro translated and35S-labeled. The control reactioncontained no plasmid DNA. The reaction products werefractionated by 11% SDS–PAGE and visualized by auto-radiography.Western blot analysispcDNA3-His, pcDSAF-1 and pCDSAF-3 plasmid-trans-fected cells were lysed in 50 mm Tris/HCl pH 7.5, 100 mmNaCl, 0.5 mm dithiothreitol, 1% Nonidet P-40, 0.1% SDS,1mm phenylmethanesulfonyl fluoride and 0.5 mgÆmL)1ofbenzamidine buffer, followed by mild sonication. Theextracts (50 lg protein) were fractionated by 11% SDS–PAGE and transferred to a nitrocellulose membrane. Toevaluate the relative amount of proteins in each lane, pro-teins were stained with Ponceau S solution (Sigma-Aldrich,St Louis, MO, USA). Immunoblotting was performed usinga 1 : 5000 dilution of anti-His tag IgG (Millipore Corpora-tion, Billerica, MA, USA). Bands were detected using achemiluminescence detection kit (Amersham Biosciences,Piscataway, NJ, USA). In some Western blots, anti-SAF-3serum was used to detect the SAF-3 protein level in IL-1b-induced or uninduced cells. SAF-3 antibody was preparedagainst the N-terminal epitope (MDPSNWSSFIFQ peptide)that is absent in the SAF-1 and SAF-2 isoforms.AcknowledgementsThis study was supported in part by US Public HealthService grants AR48762 and DK49205 and funds fromthe College of Veterinary Medicine, University ofMissouri.References1 Ray A & Ray BK (1998) Isolation and functional char-acterization of cDNA of serum amyloid A-activatingfactor that binds to the serum amyloid A promoter.Mol Cell Biol 18, 7327–7335.2 Bossone SA, Asselin C, Patel AJ & Marcu KB (1992)MAZ, a zinc finger protein, binds to c-MYC and C2gene sequences regulating transcriptional initiationand termination. Proc Natl Acad Sci U S A 89,7452–7456.3 Kennedy GC & Rutter WJ (1992) Pur-1, a zinc-fingerprotein that binds to purine-rich sequences, transacti-vates an insulin promoter in heterologous cells. ProcNatl Acad Sci U S A 89, 11498–11502.4 Ray A & Ray BK (1996) A novel cis-acting element isessential for cytokine-mediated transcriptional inductionof the serum amyloid A gene in nonhepatic cells. MolCell Biol 16, 1584–1594.5 Ray BK, Shakya A & Ray A (2007) Vascular endothe-lial growth factor expression in arthritic joint is regu-lated by SAF-1 transcription factor. J Immunol 178,1774–1782.6 Ray A, Shakya A, Kumar D & Ray BK (2004) Overex-pression of serum amyloid A activating factor 1 inhibitscell proliferation by the induction of cyclin-dependentprotein kinase inhibitor p21WAF-1/Cip-1/Sdi-1 expres-sion. J Immunol 172, 5006–5015.7 Ray A, Kuroki K, Cook JL, Bal BS, Kenter K, Aust G& Ray BK (2003) Induction of matrix metalloproteinase1 gene expression is regulated by inflammation-respon-sive transcription factor SAF-1 in osteoarthritis.Arthritis Rheum 48, 134–145.8 Ray BK, Shakya A, Turk JR, Apte SS & Ray A (2004)Induction of the MMP-14 gene in macrophages of theatherosclerotic plaque: role of SAF-1 in the inductionprocess. Circ Res 95, 1082–1090.9 Ray A, Shakya A & Ray BK (2005) Inflammation-responsive transcription factors SAF-1 and c-Jun/c-Fospromote canine MMP-1 gene expression. BiochimBiophys Acta 1732, 53–61.10 Ray A, Bal BS & Ray BK (2005) Transcriptional induc-tion of matrix metalloproteinase-9 in the chondrocyteand synoviocyte cells is regulated via a novel mecha-nism: evidence for functional cooperation betweenserum amyloid A-activating factor-1 and AP-1. J Immu-nol 175, 4039–4048.A. Ray et al. Transcription factor SAF-3 is expressed during inflammationFEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation ª 2009 FEBS 4285[...]... skeletal and cardiac myocytes Mol Cell Biol 28, 6521–6535 4286 A Ray et al 23 Ray B K, Murphy R, Ray P & Ray A (2002) SAF-2, a splice variant of SAF-1, acts as a negative regulator of transcription J Biol Chem 277, 46822–46830 24 Song J, Murakami H, Tsutsui H, Tang X, Matsumura M, Itakura K, Kanazawa I, Sun K & Yokoyama KK (1998) Genomic organization and expression of a human gene for Myc-associated zinc... Biochemistry 36, 4662–4668 15 Ray BK, Chatterjee S & Ray A (1999) Mechanism of minimally modified LDL-mediated induction of serum amyloid A gene in monocyte/macrophage cells DNA Cell Biol 18, 65–73 16 Ray A, Yu GY & Ray BK (2002) Cytokine-responsive induction of SAF-1 activity is mediated by a mitogenactivated protein kinase signaling pathway Mol Cell Biol 22, 1027–1035 17 Ray A, Ray P, Guthrie N, Shakya A, ... A, Kumar D & Ray BK (2003) Protein kinase A signaling pathway regulates transcriptional activity of SAF-1 by unmasking its DNA-binding domains J Biol Chem 278, 22586–22595 18 Kovacevic A, Hammer A, Stadelmeyer E, Windischhofer W, Sundl M, Ray A, Schweighofer N, Friedl G, Windhager R, Sattler W et al (2008) Expression of serum amyloid A transcripts in human bone tissues, differentiated osteoblast-like...Transcription factor SAF-3 is expressed during inflammation 11 Parks CL & Shenk T (1996) The serotonin 1a receptor gene contains a TATA-less promoter that responds to MAZ and Sp1 J Biol Chem 271, 4417–4430 12 Ray A, Schatten H & Ray BK (1999) Activation of Sp1 and its functional co-operation with serum amyloid A- activating sequence binding factor in synoviocyte cells trigger synergistic action of interleukin-1... method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction Anal Biochem 162, 156–159 37 Imai K, Morikawa M, D’Armiento J, Matsumoto H, Komiya K & Okada Y (2006) Differential expression of WNTs and FRPs in the synovium of rheumatoid arthritis and osteoarthritis Biochem Biophys Res Commun 345, 1615–1620 FEBS Journal 276 (2009) 4276–4286 ª 2009 The Authors Journal compilation... leukemia Leukemia 12, 326–332 21 Greiner J, Ringhoffer M, Simikopinko O, Szmaragowska A, Huebsch S, Maurer U, Bergmann L & Schmitt M (2000) Simultaneous expression of different immunogenic antigens in acute myeloid leukemia Exp Hematol 28, 1413–1422 22 Himeda CL, Ranish JA & Hauschka SD (2008) Quantitative proteomic identification of MAZ as a transcriptional regulator of muscle-specific genes in skeletal and... Eisenman RN & Bird A (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex Nature 393, 386–389 31 Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J & Wolffe AP (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription Nat Genet 19, 187–191 32 Bird A (2002) DNA methylation patterns and... interleukin-1 and interleukin-6 in serum amyloid A gene expression J Biol Chem 274, 4300–4308 13 Ray A, Fields AP & Ray BK (2000) Activation of transcription factor SAF involves its phosphorylation by protein kinase C J Biol Chem 275, 39727–39733 14 Ray BK & Ray A (1997) Involvement of an SAF-like transcription factor in the activation of serum amyloid A gene in monocyte/macrophage cells by lipopolysaccharide... protein (MAZ) J Biol Chem 273, 20603–20614 25 Kozak M (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes Cell 44, 283–292 26 Ray A, Kumar D, Ray P & Ray BK (2004) Transcriptional activity of SAF-1 is regulated by distinct functional modules J Biol Chem 279, 54637–54646 27 Hanke J, Brett D, Zastrow I, Aydin A, Delbruck S, Lehmann G,... 6–21 33 Miranda TB & Jones PA (2007) DNA methylation: the nuts and bolts of repression J Cell Physiol 213, 384–390 34 Baylin SB (2002) Mechanisms underlying epigenetically mediated gene silencing in cancer Semin Cancer Biol 12, 331–337 35 Sambrook J & Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 36 Chomczynski P & Sacchi N . SAF-3, a novel splice variant of the SAF-1/MAZ/Pur-1 family, is expressed during inflammation Alpana Ray1, Srijita Dhar1, Arvind Shakya1, Papiya. CTTCTCCCGgtgtgcac 403 gtccccagGCCGGATCA364AATGTGAGgtaggaag 277 ctcctcagAAATGTGAG4 172 CAACAAAGgtacatgc 1335 ctgtgcagGTACTGGTG5 1028 A. Ray et al. Transcription factor
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